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| Status |
Public on Nov 28, 2023 |
| Title |
Rat5 day14 ipsilateral |
| Sample type |
RNA |
| |
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| Source name |
ipsilateral
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
tissue: spinal cord - C5 to T1 spinal segments
gender: female
weight: 180-250g
treatment: 14 days after the unilateral BPRA in the spinal cord
|
| Treatment protocol |
In the supine position, the right brachial plexus was exposed, and the right C5-C8 and T1 spinal nerve roots were isolated under a surgical microscope (Chenghe Microsurgical Instruments Factory, China)，and then were pull out one by one using micro hemostatic forceps. The avulsed ventral and dorsal roots together with the dorsal root ganglia were cut away from the distal ends of the spinal nerves and success of avulsion model confirmed under the microscope. The muscle and skin were sutured in successive layers following avulsion. The animals were placed in a heated recovery chamber and returned to their cage once they were awake.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The rats were sacrificed on the 3rd day or the 14th day after root avulsion. They were given a lethal dose of 10% chloral hydrate (Kermel, China). Under the surgical microscope, the cervical spinal cord of each rat was exposed, and the ipsilateral half of the spinal cord segments with a complete ventral and dorsal root avulsion were evaluated and success of avulsion confirmed. Then, the C5 to T1 spinal segments were removed and dissected into ipsilateral and contralateral halves as quickly as possible. Total RNA was isolated using TRIzol (TIANGEN, China) according to the manufacturer's instructions. Total RNA from each sample was quantified using a Nanodrop 2000 (Thermo fisher, US), and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. Subsequently, we stored the remaining RNA at − 80 °C for later use.
|
| Label |
Cy3
|
| Label protocol |
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a mixture of oligo(dT) and random primers (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
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| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65 °C in an Agilent Hybridization Oven.
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| Scan protocol |
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 3 out of 6 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs with statistical significance between the two groups were identified through P-value/FDR filtering. Differentially expressed LncRNAs and mRNAs between the two samples were identified through Fold Change filtering. Hierarchical Clustering was performed using homemade scripts.
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| |
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| Submission date |
Nov 27, 2018 |
| Last update date |
Nov 28, 2023 |
| Contact name |
Guangyin Yu |
| E-mail(s) |
yugy6@mail2.sysu.edu.cn
|
| Organization name |
Zhongshan School of Medicine, Sun Yat-sen University
|
| Street address |
No. 74 Zhongshan Road 2, Guangzhou 510080, P.R. China
|
| City |
Guangzhou |
| ZIP/Postal code |
510080 |
| Country |
China |
| |
|
| Platform ID |
GPL15690 |
| Series (1) |
| GSE123007 |
The nerve roots avulsion induced time-specific expression patterns of lncRNA and mRNA occurred before the motoneurons loss of the injured spinal segments |
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